From article- Purification and crystallization of the catalytic domain of human protein tyrosine phosphatase 1B expressed in Escherichia coli
“…PTP1B was eluted by applying a linear gradient of 30ml to buffer C + 10% (v/v) glycerol. The protein, at greater than 99% homogeneity, was dialysed against 10 mM Tris HCL (pH7.5), 25mM NaCl, 0.2mM EDTA and 3 mM dithiothreitol, concentrated to 10mg/ml and either used for crystallization or stored in 30% (v/v) glycerol at -20°C.”
From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28320/?tool=pubmed "PTP1B/C215S was eluted from the column by a linear gradient from 0 to 0.5 M NaCl in 200 ml of buffer A.” buffer A: [100 mM 2-(4-morpholino)-ethane sulfonic acid, pH 6.5/1 mM EDTA/1 mM DTT]
Crystallization Conditions (list source of information - e.g. PDB entry, BRENDA, specific reference to a paper, etc.):
From same article (Purification and ...) as above:
“Crystals were grown using the vapor diffussion method at 4°C. PTP1B (5 ul of 10mg/ml) was mixed with an equal volume of precipitating solution, 0.1 M Hepes (pH 7.5), 0.2 M magnesium acetate, 12% to 16% (w/v) polyethylene glycol 8000 (Fluka), and applied to a silanized glass cover-slop inverted over a well solution containing 0.7 ml of the preciptating solution in tissue culture trays (Linbro). Single crystals exhibiting a trigonal morphology appear within a few days and reach a maximum size of 0.1 mm x 0.2 mm x 0.2 mm within a week.”
“Crystals were incubated in a cryoprotectant buffer consisting of precipitating solution with glycerol. The glycerol was brought to 25% (v/v) in five percent steps over a period of two hours, after which the crystals were frozen in the nitrogen stream produced by an Oxford Cryostream cooling device operating at 100 K, before being transferred to liquid nitrogen for long term storage.”
"Crystals were grown using sitting drop vapor diffusion at 4 °C by mixing 2 μl of protein solution, 0.5 μl of 30% (w/v) sucrose, and 3 μl of precipitant solution (0.1 mHepes, pH 7.5, 0.2 m magnesium acetate, and 15–17% polyethylene glycol 8000). Single crystals appeared after 3 days. The protein solution was prepared as follows. 0.36 μl of 100 mm of Na3VO4 and 10 μl of 50 mm DADEYL peptide (at pH 8.5–9.0) were mixed and allowed to react for 1–1.5 h; 50 μl of native PTP1B (12 mg/ml in 10 mm Tris, pH 7.5, 25 mm NaCl, 0.2 mm EDTA, and 3 mm dithiothreitol) was then added to the peptide/vanadate mixture and immediately placed in the crystallization tray. The final protein solution contained a ratio of 1:3:30 between PTP1B, Na3VO4, and the peptide DADEYL. Crystallization at higher concentrations of Na3VO4 resulted in different crystal morphology; a 30:3 vanadate/peptide ratio gave crystals that proved to be a binary complex between PTP1B and VO43−. Cryoprotection was performed by transferring crystals stepwise into stabilization solution with increasing glycerol amounts to a final concentration of 15% and the respective initial concentrations of ligands present in the protein solution, and then flash-cooled in liquid nitrogen."
From Crystal structure of ED-Eya2: insight into dual roles as a protein tyrosine phosphatase and a transcription factor
A full-length clone of human Eya2 (gene accession no. BC008803; OpenBiosystems, Huntsville, AL, USA) was subcloned into pET28a. ED-Eya2 (residues 268–538) was expressed in the BL21 (DE3) strain of Escherichia coli. Cells were grown at 295 K after induction with 0.1 mM IPTG for 20 h. Cells were harvested and suspended in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM PMSF, 0.05% (v/v) 2-mercaptoethanol, and 5% (v/v) glycerol. After lysing cells by sonication, His-tagged ED-Eya2 was purified using nickel-affinity chromatography, and the histidine-tag was removed by thrombin protease digestion. ED-Eya2 was purified further by DEAE-Sepharose FF ion exchange chromatography and gel filtration chromatography, then equilibrated with buffer containing 20 mM HEPES–NaOH (pH 7.0), 0.2 M NaCl, 5 mM MgCl2, 2 mM DTT, and 5% glycerol. The catalytic activity of ED-Eya2 was determined using a spectrofluorometric assay (see Supplemental Data). To prevent protein oxidation, 20 mM DTT was added, and the protein was concentrated to 18 mg/ml for crystallization."
plates with pNPP soaked in at around 10 mM with PTP1b
plates with HTS09350SC soaked in at 3-5 times Ki.
Inhibitor from Spring 2011 class that showed promise - originally picked by Radhika from her screening in GOLD
HTS09305SC from Hit Finder 9 library http://www.maybridge.com/portal/alias__Rainbow/lang__en/tabID__191/DesktopDefault.aspx
Ran search in Binding DB for similar hits - no exact matches found. Closest ones with Tanimoto Score of 0.7 or greater were beta-lactamase inhibitors.
Need roughly 3-5 times the Ki if want to soak in with crystals.
Can determine Ki of inhibitor from IC50 and Km of substrate when using Cheng-Prusoff equation
Ki = (IC50)/ (1+([S]]/Km))
SUBSTRATE pNPP Km is 0.2 - 2.5 mM
guess IC50 as 0.400 mM for inhibitor HTS09350SC
Storage Conditions (list source of information - e.g. PDB entry, BRENDA, specific reference to a paper, etc.):
080811 - DTT in storage buffer is key to preventing oxidation of active site Cysteine. TCEP would be an even better reducing agent.
From Brenda:
From article- Purification and crystallization of the catalytic domain of human protein tyrosine phosphatase 1B expressed in Escherichia coli
“…PTP1B was eluted by applying a linear gradient of 30ml to buffer C + 10% (v/v) glycerol. The protein, at greater than 99% homogeneity, was dialysed against 10 mM Tris HCL (pH7.5), 25mM NaCl, 0.2mM EDTA and 3 mM dithiothreitol, concentrated to 10mg/ml and either used for crystallization or stored in 30% (v/v) glycerol at -20°C.”
From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28320/?tool=pubmed
"PTP1B/C215S was eluted from the column by a linear gradient from 0 to 0.5 M NaCl in 200 ml of buffer A.” buffer A: [100 mM 2-(4-morpholino)-ethane sulfonic acid, pH 6.5/1 mM EDTA/1 mM DTT]
Crystallization Conditions (list source of information - e.g. PDB entry, BRENDA, specific reference to a paper, etc.):
From same article (Purification and ...) as above:
“Crystals were grown using the vapor diffussion method at 4°C. PTP1B (5 ul of 10mg/ml) was mixed with an equal volume of precipitating solution, 0.1 M Hepes (pH 7.5), 0.2 M magnesium acetate, 12% to 16% (w/v) polyethylene glycol 8000 (Fluka), and applied to a silanized glass cover-slop inverted over a well solution containing 0.7 ml of the preciptating solution in tissue culture trays (Linbro). Single crystals exhibiting a trigonal morphology appear within a few days and reach a maximum size of 0.1 mm x 0.2 mm x 0.2 mm within a week.”
“Crystals were incubated in a cryoprotectant buffer consisting of precipitating solution with glycerol. The glycerol was brought to 25% (v/v) in five percent steps over a period of two hours, after which the crystals were frozen in the nitrogen stream produced by an Oxford Cryostream cooling device operating at 100 K, before being transferred to liquid nitrogen for long term storage.”
From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2871455/?tool=pubmed
"Crystals were grown using sitting drop vapor diffusion at 4 °C by mixing 2 μl of protein solution, 0.5 μl of 30% (w/v) sucrose, and 3 μl of precipitant solution (0.1 mHepes, pH 7.5, 0.2 m magnesium acetate, and 15–17% polyethylene glycol 8000). Single crystals appeared after 3 days. The protein solution was prepared as follows. 0.36 μl of 100 mm of Na3VO4 and 10 μl of 50 mm DADEYL peptide (at pH 8.5–9.0) were mixed and allowed to react for 1–1.5 h; 50 μl of native PTP1B (12 mg/ml in 10 mm Tris, pH 7.5, 25 mm NaCl, 0.2 mm EDTA, and 3 mm dithiothreitol) was then added to the peptide/vanadate mixture and immediately placed in the crystallization tray. The final protein solution contained a ratio of 1:3:30 between PTP1B, Na3VO4, and the peptide DADEYL. Crystallization at higher concentrations of Na3VO4 resulted in different crystal morphology; a 30:3 vanadate/peptide ratio gave crystals that proved to be a binary complex between PTP1B and VO43−. Cryoprotection was performed by transferring crystals stepwise into stabilization solution with increasing glycerol amounts to a final concentration of 15% and the respective initial concentrations of ligands present in the protein solution, and then flash-cooled in liquid nitrogen."
From Crystal structure of ED-Eya2: insight into dual roles as a protein tyrosine phosphatase and a transcription factor
http://www.fasebj.org/content/24/2/560.long
* Not sure if this is for PTP1B in homo sapiens*"Expression and purification
A full-length clone of human Eya2 (gene accession no. BC008803; OpenBiosystems, Huntsville, AL, USA) was subcloned into pET28a. ED-Eya2 (residues 268–538) was expressed in the BL21 (DE3) strain of Escherichia coli. Cells were grown at 295 K after induction with 0.1 mM IPTG for 20 h. Cells were harvested and suspended in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM PMSF, 0.05% (v/v) 2-mercaptoethanol, and 5% (v/v) glycerol. After lysing cells by sonication, His-tagged ED-Eya2 was purified using nickel-affinity chromatography, and the histidine-tag was removed by thrombin protease digestion. ED-Eya2 was purified further by DEAE-Sepharose FF ion exchange chromatography and gel filtration chromatography, then equilibrated with buffer containing 20 mM HEPES–NaOH (pH 7.0), 0.2 M NaCl, 5 mM MgCl2, 2 mM DTT, and 5% glycerol. The catalytic activity of ED-Eya2 was determined using a spectrofluorometric assay (see Supplemental Data). To prevent protein oxidation, 20 mM DTT was added, and the protein was concentrated to 18 mg/ml for crystallization."
=
For crystallization:can use our Homebrew Crystal Conditions ( % NaC, Na Acetate, pH, ethylene glycol) screen, Hampton Crystal Screen or Hampton Crystal Screen 2
Inhibitor from Spring 2011 class that showed promise - originally picked by Radhika from her screening in GOLD
HTS09305SC from Hit Finder 9 library
http://www.maybridge.com/portal/alias__Rainbow/lang__en/tabID__191/DesktopDefault.aspx
Ran search in Binding DB for similar hits - no exact matches found. Closest ones with Tanimoto Score of 0.7 or greater were beta-lactamase inhibitors.
Need roughly 3-5 times the Ki if want to soak in with crystals.
Can determine Ki of inhibitor from IC50 and Km of substrate when using Cheng-Prusoff equation
Ki = (IC50)/ (1+([S]]/Km))
SUBSTRATE pNPP Km is 0.2 - 2.5 mM
guess IC50 as 0.400 mM for inhibitor HTS09350SC